Storage and maintenance of platelets

ABSTRACT

Cell membrane maintenance of red blood cells and platelet concentrates is improved by the addition of 1 mM-10 mM L-carnitine and derivatives. This improvement allows extension of the period of viability of packed red blood cells and platelet concentrations beyond current periods. Additionally, the materials so treated exhibit extended circulation half life upon transfusion to a patient. Improvements in membrane maintenance achieved by this method permit irradiation of sealed containers of blood products so as to substantially sterilize and destroy leukocytes in the same.

The present application is a Continuation application of U.S. Ser. No.08/840,765 filed on Apr. 16, 1997, now U.S. Pat. No. 6,482,585.

FIELD OF THE INVENTION

This invention pertains to a method of improving the storage stability,including resistance to hemolysis and improved viability, of bloodproducts including packed red blood cells (RBCs), platelets and thelike. Specifically, a method for extending the viability of theseproducts, as well as their resistance to membrane damaging agents suchas radiation, is provided by storing the products in a suspensionincluding an effective amount of L-carnitine or alkanoyl carnitines.

BACKGROUND OF THE INVENTION

Concern has been steadily growing over both the national, and worldwideblood supplies. Both the integrity and reliability of existing supplies,and the ability to build larger stocks over time, has been brought intoquestion. One reason for this is the relatively short period of storagestability of blood products. Currently, packed RBCs (red blood cellconcentrates, or RCC), the dominant form of blood product fortransfusions and the like, are limited to a 42-day storage period. Afterthat time, ATP levels fall substantially, coupled with a significantloss of pH, strongly indicating a lack of viability, or, if viable, anextremely short circulation life upon infusion, in vivo whole blood isnot stored for substantial periods. For platelets, the current storageperiod is even shorter, with the standard being 5-days at 22° C. Thedifference in storage stability of platelet concentrations (PC) hasopposed to RBC, is due to ongoing metabolic reactions in patelets, duein part the presence of mitochondria in PC, and their absence in RBCs.While both blood products show a drop in ATP, coupled with a drop in pH,over time, accompanied by the production of lactic acid, the presence ofmitochondria in PC is likely to exacerbate the problem, due toglycolysis.

Simultaneously, concerns over the reliability and integrity of the bloodsupply have been raised. In particular, contamination of the bloodsupply with bacteria, or other microbiological agents, has been detectedrepeatedly. Such a situation is even more severe in countries with lesssophisticated collection and storage methods. While agents may be addedto collected products to reduce contamination, these are not desirable,given the need to transfuse the products back into recipient patients.One desirable alternative is radiation treatment of the products, afterpackaging, typically in plasticised vinyl plastic containers. Suchradiation treatment would aggravate RBC and perhaps during PC storage,resulting in a diminished function of these cells.

Additionally, a small but growing portion of the blood receivingpopulation is at risk of a generally fatal condition known asTransfusion associated graft versus host disease (TA-GVHD), which is duethe presence of viable allogenic leukocytes. This syndrome is typicallyassociated with immunosuppressed patients, such as cancer and bonemarrow transplant patients, but can also occur in immunocompetentpersons in the setting of restricted HLA polymorphism in the population.

Substantial attention has been devoted to finding methods to extendstorage stability. One such method, for extending the storage lifetimeof PCs, is recited in U.S. Pat. No. 5,466,573. This patent is directedto providing PC preparations with acetate ion sources, which acts bothas a substrate for oxidative phosphorylation and as a buffer tocounteract pH decrease due to lactic acid production. Such a method doesnot act directly on the problem of hemolysis, and membrane breakdown. Analternative method is disclosed in U.S. Pat. No. 5,496,821, by theinventor herein and commonly assigned. In this patent, whole blood isstored in a preparation including L-carnitine (LC) or alkanoylderivatives thereof. The patent does not describe, however, the effectson blood products such as PC or RBC suspensions, and relies to at leastsome extent on the impact of LC on plasma characteristics.

As noted above, contamination of the blood supply with microbiologicalagents is another problem to be addressed by the medical community. Onemethod of sterilizing the product, and improving reliability withrespect to contamination, is to irradiate the blood product. In general,gamma irradiation values of about 25 centigray (cG), irradiating theproduct after it is sealed in a plastic, glass or other container isdesirable. Regrettably, irradiation induces cell membrane lesions, withhemolysis in RBCs. Irradiation of blood products, including whole blood,packed RBCs and PCs continue to pose problems.

Accordingly, it remains an object of those of skill in the art toprovide a method to extend the period of viability, and the circulationhalf-life of RBCs and PCs upon transfusion, beyond the current maximums.Additionally, it remains a goal of those of skill in the art to find away by which blood products, including whole blood, packed RBCs and PCscan be sterilized by irradiation, without substantial membrane damageand lesions, and hemolysis.

SUMMARY OF THE INVENTION

The Applicant has discovered, through extended research, that themembrane damage experienced by RBCs and PCs upon storage, or in the faceof irradiation, can be substantially delayed and suppressed, bysuspending the blood product in a conventional preservation solution,such as AS-3, where the preservation solution further includesL-carnitine or an alkanoyl derivative thereof, in a concentration of0.25-50 mM or more. Applicant's discovery lies in the recognition thatmost of the decomposition of blood products, conventionally associatedwith decreases in ATP levels, and pH, can be in fact traced to membranedamage and hemolysis. Membrane maintenance and repair may be effected bylipid reacylation, effected, in part, through LC, the irreversibleuptake of which in RBC and similar blood products has been establishedthrough the inventive research.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Lifespan values after infusion of 42 days stored rbC as relatedto donor and to control and LC stored. The arrows indicate mean±SD ofcontrol and LC stored RBC, respectively. On top of the graph, the exactcalculated p value is also shown.

FIG. 2. Red cell carnitine content at different weeks of bloodpreservation. Carnitine was assayed as described in Materials andMethods. Values are the average of three experiments done in duplicate.The variation between experiments was not more than 7%. Open symbols,RBC stored with AS-3 alone; closed symbols, RBC stored in AS-3supplemented with LC (5 mM).

FIG. 3. Radioactive palmitic acid incorporation into RBC LC at differentweeks of blood preservation. RBC aliquots withdrawn either from bloodunit stored in AS-3 alone or AS-3 plus LC were incubated at 37° C. with[1-¹⁴C]palmitic acid complexed to fatty acid free BSA. At the end ofincubation, RBC were then processed as described in Materials andMethods. Radiolabeled PLC formation was referred to the phosphorouscontent present in lipid extract. Values are the average of threeexperiments done in duplicate. The variation between experiment was notmore than 7%. Open symbols, RBC stored with AS-3 alone; closed symbols,RBC stored in AS-3 supplemented with LC (5 mM).

FIG. 4. Radioactive palmitic acid incoropration into red cell membranePE and PC at different weeks of blood preservation. RBC aliquotswithdrawn either from blood unit stored in AS-3 alone or AS-3 plus LCwere incubated at 37° C. with [1-¹⁴C]palmitic acid complexed to fattyacid free BSA. At the end of incubation, RBC were then processed asdescribed. Results are given as pmol [1-¹⁴C]palmitic acid/μg lipidphosphorous present in lipid extract. Values are the average of threeexperiments done in duplicate. The variation between experiments was notmore than 7%. Open symbols, RBC stored with AS-3 alone, closed symbols,RBC stored in AS-3 supplemented with LC (5 mM).

FIG. 5. The carnitine system and membrane phospholipid reacylationreactions. Thicker arrows indicate the preferential acyl flux. Thedimension of the acylcarnitine box shows the likely related pool size.Abbreviations used are: LPL, lysophospholipids; PLP, phospholipids; Cn,carnitine; acyl-Cn, acyl-carnitine; ACS, acyl-CoA synthetase; LAT,lysophospholipid acyl-CoA transferase; CPT, carnitinepalmitoyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

This invention employs L-carnitine, and its alkanoyl derivatives, as anagent supporting cell membrane maintenance and repair, and suppressionof hemolysis, in blood products. Alkanoyl L-carnitines includes acetyl,butyryl, isobutyryl, valeryl, isovaleryl and particularly propionylL-carnitine. Herein, reference is made to this family, generically, asLC, and exemplification is in terms of L-carnitine. The describedalkanoyl L-carnitines, and their pharmalogically acceptable salts,however, may be used in place of L-carnitine.

The addition of LC to blood products, including RBCs and PCs, requiresLC to be present in an amount effective to permit membrane maintenance,repair and hemolysis suppression. The research undertaken, including theexamples set forth below, has demonstrated a minimum effective range forthe products of most donors of about 0.25 mM-0.5 mM. The upper limit ismore practical than physiological. Concentration as high as 50 mM orgreater are easily tolerated. Values that are consistent withtoxicological and osmological concerns are acceptable. Preferred rangesare 1-30 mM. A range of 1-10 mM or more is suitable with values between4-6 mM making a marked difference. The effects of this invention,including the prolongation of viability, and the extension ofcirculation half-life upon transfusion, may be highly donor dependent.Accordingly, generally speaking, an effective concentration of LC is0.5-50 mM, however, the ordinary artisan in the field may be required toextend that range, in either direction, depending on the particularitiesof the donor. Such extensions do not require inventive effort.

LC is consistent with conventional support solutions (stabilizingsolutions), which are typically prepared to provide a buffering effect.Commonly employed solutions include ACED (citric acid-sodiumcitrate-dextrose), CPD (citrate-phosphate-dextrose) and modificationsthereof, including CPD2/A-3, and related compositions. Typically, thecomposition includes a carbohydrate, such as glucose or mannitol, atleast one phosphate salt, a citrate, and other balancing salts. LC isconventionally soluble and may be added to these compositions freelywithin the required range. Suitable solutions, are described in U.S.Pat. No. 5,496,821, incorporated herein-by-reference. Note, however,that support solutions other than those conventionally used can beemployed, including artificial plasma and other physiologicallyacceptable solutions can be use with LC in the invention. The importantcomponent of the support solution is LC.

The ability of LC, when included in the suspension of blood productssuch as RBC and PCs, to extend the viable time and therefore shelflength, and the circulation period upon transfusion into the receivingindividual, is exemplified below by in vitro and in vivoexperimentation. The experimentation employs LC, but other alkanoylL-carnitines can be employed. Of particular significance is thedemonstration, below, that the improved performance is obtained throughimproved maintenance (including repair) of the cell membrane, andsuppression of hemolysis.

MATERIALS AND METHODS

Study Design I:

Evaluation of in Vivo and in Vitro Quality of RBC Stored with andwithout LC

Subjects. The subject population was male or female research subjectsbetween the ages of 18 to 65 years with no known mental or physicaldisability and taking no drugs that might affect RBC viability.Individuals were recruited who fulfilled the conventional allogeneicdonor criteria as listed in the Code of Federal Register, Chapter 2, theStandards of the American Association of Blood Banks, and the BloodServices Directives of the American National Red Cross. The study wasapproved by the Institutional Review Board of the Medical College ofHampton Roads and the subjects gave informed consent prior toparticipation in the study.

Each donor donated on two different occasions separated by 72 days, andwas randomized to either the test or control arm on the first donation.

Blood storage system. Standard CP2D/AS-3 system (Miles, Inc.) Usingpolyvinyl chloride (PVC) plastic with diethyl-(n)hexyl-phthalate (DEHP)as plasticizer was used. For each test unit, 245 mg LC (in 1.1 mL pure,pyrogen-free solution in a sterilized glass bottle) was added to thecontainer holding the AS-3 additive solution to give a finalconcentration of 5 mM. For the control units, 1.1 mL 0.9% NaCl was addedto the AS-3 solution using the same conditions. Addition of LC or salineto the bags was performed by injecting through a sampling site couplerwith a syringe. This was done in a laminar flow hood under UV light.Donation & Processing. Standard phlebotomy and blood-drawing methodswere used with collection of approximately 450±50 mL whole blood. Thewhole blood unit was held between 4-8 hours at room temperature beforeprocessing. The unit was centrifuged using standard conditions and,after centrifugation, the supernatant plasma was expressed off, and thesedimented packed RBC resuspended either in the standard AS-3 solution(control) or the carnitine-containing AS-3 solution (test). Thesuspended RBC units were stored at 4° C. for 42 days.In Vitro Measurements: Measurements performed on pre-(0 day) andpost-(42-day) samples included RBC ATP levels; total and supernatanthemoglobin; hematocrit (Hct); RBC, WBC, and platelet counts; RBC osmoticfragility; RBC morphology; lactate and glucose levels; supernatantpotassium levels. These were performed using standard procedures asdescribed previously, Heaton et al., Vox Sang 57:37-42 (1989).In vivo Post-transfusion Measurements. After 42 days of storage, asample was withdrawn and the stored cells labeled with Cr using standardmethods. At the same time, to determine RBC mass, a fresh sample wascollected from the donor for RBC labeling with 99 Tc. After labeling, 15μCi 51 Cr-labeled stored cells and 15 μCi 99 Tc-labeled fresh cells weremixed and simultaneously infused. Blood samples (5 mL) were taken afterthe infusion at various time intervals for up to 35 days to calculate24-hour % recovery and survival. The 24-hour % recovery was determinedusing either the single label method where log-linear regression of theradioactivity levels of samples taken at 5, 7.5, 10, and 15 min. wasused to determine 0 time level, or by the double label method usingdonor RBC mass as determined by the 99 Tc measurement.

Circulating lifespan of the transfused surviving Cr-labeled RBC wasdetermined by samples taken at 24 hours and then twice weekly for up to5 weeks. The radioactivity levels were corrected for a constant 1%elution per day. The data were fitted to a linear function withpost-transfusion days as independent variable (x-axis) and the correctedCr counts as dependent variable (y-axis). The lifespan of the RBC wasthen taken as the intersection of the fitted line with the x-axis.

Statistical analysis. Paired t-test or routine non-parametricstatistical analysis was performed on data from the in vivo and in vitrotesting of the units to determine if there were any statisticallysignificant differences (1-tail) in the means between the test andcontrol units. Statistical significance was considered at a p value lessthan 0.05.Study Design IIErythrocytes LC Uptake and Lipid Reacylation Studies with Storage up to42 DaysChemicals. Essentially fatty acid-free bovine serum albumin (BSA) wasobtained from SIGMA Chemical Company, St. Louis, Mo. (USA).[1-¹⁴C]Palmitic acid (58 Ci/mol) was obtained from New England NuclearCorporation, Boston, Mass. (USA). Thin-layer plates, Whatman LK6 (silicagel) (20×20 cm) with a pre-absorbent layer were obtained from CarloErba, Milan (Italy). Palmitoyl-L-carnitine (PLC) and LC were a kind giftof Sigma Tau, Pomezia (Italy). All other compounds used were reagentgrade.Red cell carnitine assay. Blood sample was withdrawn from the stored RCCunit and washed once with 4 vol. of cold 0.9% NaCl. RBC were thenresuspended in 0.9% NaCl at a final hematocrit of 50%, and deproteinizedwith perchloric acid as described, Cooper et al., Biochem. Biophys. Acta959: 100-105 (1988). Aliquots of the final extract were analyzed forfree LC content according to the radiochemical assay of Pace et al.,Clin. Chem. 24: 32-35 (1978).Analysis of membrane complex lipid reacylation in stored RBC. Bloodsample was withdrawn from stored RCC unit through a sampling sitecoupler with a syringe, and the sample processed immediately. This wasdone in a laminar flow hood under UV light. All the manipulations wereconducted at 0-5° C. unless noted. RBC were washed two times with 4 vol.of cold 0.9% NaCl. Isolated RBC were once again washed with incubationbuffer (NaCl 120 mM, KCl 5 mM, MgSO₄ 1 mM, NaH₂PO₄ 1 mM, saccharose 40mM, 5 mM glucose, Tris-HCl 10 mM, at pH 7.4) and resuspended in the samebuffer at a final hematocrit of 5%. A Rotabath shaking bath at 37° C.was used for the incubations. RBC were incubated with the radioactivepalmitic acid (10 μM) complexed to fatty acid free BSA (1.65 mg/ml).Incubations were ended by washing cells once with cold incubationbuffer, three times with fatty acid free BSA 1% in incubation buffer,and finally once again with incubation buffer. RBC lipids were extractedfrom intact cells with the Rose & Oaklander procedure, J. Lipid Res. 6:428-431 (1965). In order to prevent lipid oxidation, 0.1% butylatedhydroxytoluene was added to the lipid extracts. Aliquots of the lipidextract were used for determination of lipid phosphorus content, andanalyzed by two dimensional thin layer chromatography. Briefly, thechromatograms were developed using chloroform-methanol-28% ammonia(65:25:5) in the first dimension. The chromatograms were then developedusing chloroform-acetone-methanol-acetic acid-water (6:8:2:2:1) in thesecond dimension. Phosphatidylcholine (PC), a phosphatidylethanolamine(PE), and phosphatidylserine were visualized by brief exposure of theplates to iodine and identified using standards as a reference.Individual phospholipid spots were scraped off into vials containingscintillation fluid and radioactivity was determined by liquidscintillation counting. The identification and analysis of radioactivePLC was carried out as recently described, Arduini et al., J. Biol.Chem. 267: 12673-81 (1992). Counting efficiency was evaluated by anexternal standard. Calculations are based on the specific activities ofradioactive palmitic acid.

Results

Study I

Pre-Storage AS-3 RCC Unit Characteristics

The properties of the AS-3 RCC products were as expected after theprocessing of the whole blood units. No significant difference betweentest and control units in the characteristics of the AS-3 RBC unit wereobserved as measured by unit volume, Hct, and WBC content, and in vitroRBC properties such as ATP levels, supernatant hemoglobin and potassiumlevels, osmotic fragility (Table I).Post-42 Day Storage RBC CharacteristicsMetabolic. The amounts of glucose consumed and lactate produced during42 days of storage were similar for test and control units (Table 2). Asexpected, an inverse high correlation was found between these twoparameters of glycolysis (r=0.76). However, less hemolysis and higherATP levels were found for carnitine-stored RBC units as compared tocontrol. As illustrated in FIG. 1, this higher ATP level was observed inall but one pair (p<0.01).Membrane. Percent hemolysis at the end of storage levels was less withthe carnitine units, as shown in FIG. 2. On the other hand, nosignificant differences were found with regard to supernatant potassiumlevels, osmotic shock response, and morphology score which were withinexpected range at the end of 42 days of storage.Post transfusion viability. The mean 24-hour % recovery for the controlunits was similar to what previously has been found by us and by others.However, mean % recoveries for the carnitine-stored red cells werehigher than the control-stored cells (p<0.05). In addition, the meancirculating lifespan of the infused stored red cells was also higher forthe carnitine-stored cells (FIG. 1). The donors' RBC mass as determinedon the two occasions were highly similar (r=0.98) and statistically notdifferent.Correlation studies. As expected, 24-hour % recovery showed significantcorrelations with ATP levels (r=0.63) and other measurements of RBCmembrane integrity such as hemolysis (r=0.57), osmotic fragility(r=0.71), and morphology score (r=0.59). Percent hemolysis correlatedhighly with ATP levels (r=0.83) and also with the WBC content of the RBCunits (r=0.83). The RBC circulating lifespan showed no significantcorrelations with any in vitro parameter.Study IICarnitine Uptake in Stored RBC

RBC stored in AS-3 medium alone did not show any significant loss of theLC content throughout the storage (FIG. 2). This is in agreement withfindings by Cooper et al., supra showing that human red cell LC does notfreely exchange with either plasma or isoosmotic buffer. When red cellswere stored in AS-3 supplemented with LC, higher amounts ofintracellular LC than AS-3 alone were detected (FIG. 2). LC contentincreased linearly during times of storage, reaching a 4 fold increaseat 42 days.

Membrane Complex Lipid Reacylation Studies in Stored RBC

With no carnitine present the radioactive palmitate incorporated intoPLC decreased linearly with the time of storage (FIG. 3). At variance,red cell from RCC unit stored in the LC-containing the AS-3 solutionshowed an initial increase (with a nadir at the 3 week) followed by arapid decrease of the radioactive palmitate incorporated into PLC. Inthe same red cell preparations, the incorporation of radioactivepalmitate into membrane phospholipids was also evaluated. Radioactivepalmitate incorporation into membrane PE of red cells from blood unitstored in the AS-3 solution alone showed a constant significant increaseof radioactivity into PE throughout the storage period (FIG. 4). Redcells stored in the presence of LC were characterized by a suddenincrease of PE reacylation toward the end of the storage, with a nadirat the 6^(th) week (FIG. 4). Radioactive palmitate incorporation ratesinto membrane PC decreased slightly throughout the storage, and nodifferences were observed between the two red cell preparations (FIG.4). It should be pointed out that since in our reacylation studies redcells were incubated at 37° C. in a Krebs Ringer buffer containingglucose, ATP levels at the end of the incubation were close tophysiological values (data not shown).

Discussion

In this study, in vitro and in vivo testing of RBC units at the end of42 days of storage demonstrated significant differences betweencarnitine-stored RBC as compared to control-stored RBC. Various in vitroRBC properties reflective of metabolic and membrane integrity such asATP and % hemolysis, as well as direct measure of cell viability(24-hour % recovery and circulating lifespan) were significantlysuperior for carnitine-stored RBC. A prolongation of the mean lifespanof the surviving RBC circulating at 24-hours after infusion is ofinterest. This finding may be related to the irreversible uptake of LCduring storage an unprecedented and unexpected discovery. The valuesobtained in the control studies for various RBC properties were asexpected and not different from previous studies. At the time of bloodcollection, no significant differences in unit or RBC characteristicsbetween test and control were found. As illustrated in FIGS. 1-3, RBCATP levels, % hemolysis, and circulating lifespan were stronglydonor-related, and, since the study was a randomly paired design withfive test and five control studies performed on both the first andsecond occasions, it is unlikely that the observed differences could bedue to chance or to any faulty study design. It is, therefore, mostlikely that the observed differences found in this study were caused bythe addition of carnitine to the test units. The possibility that theincreased lifespan reflects decreased elution of Cr cannot be excluded,but is not consistent with the improved in vitro measures of the storedred cells that has been found to correlate with in vivo viability.

Several investigations have found that LC and its acyl-esters have acytoprotective/membrane stabilizing effect on various cells includingred cells. See, e.g., Snyder et al., Arch. Biochem. Biophys. 276:132-138 (1990). In this study it was found that LC was irreversiblytaken up by the RBC during storage. Although the nature of this processis not entirely clear, one would exclude the participation of a specificcarrier for the LC uptake. To our knowledge, the only known LC carrieroperates in cellular systems where the intracellular concentration of LCis several fold higher than that normally present in the extracellularenvironment. Red cell LC concentration is similar to that of the plasma.Thus, irrespective of the low temperature, APT depletion, and otherpossible metabolic changes occurring during the storage, when red cellsare stored in a medium containing relatively high amounts of exogenousLC, a unidirectional uptake of LC by the cells seems to be established.Nothing in the art appears to predict this.

The nature of the action of LC on stored RBC could be viewed either as abiophysical and/or metabolic intervention on the membrane compartment.Post-transfusion survival of stored red cells is related to theintegrity of membrane function as suggested by the significantcorrelation between the in vivo viability of reinfused red cell and itssurface-to-volume ratio measures. A major contributor to RBC membranestructure and function is represented by the cytoskeleton network,Marchesi, Ann. Rev. Cell Biol. 1: 531-536 (1985), a supramolecularprotein organization lying beneath the inner hemileaflet of RBCmembrane. Wolfe et al in a survey study on the composition and functionof cytoskeletal membrane protein of stored red cells found that the onlyrelevant change was a decreased capability of spectrin to associate withactin either in the presence or absence of protein 4.1. Wolfe et al., J.Clin. Invest. 78: 1681-1686 (1986). We have shown that LC affect RBCmembrane deformability of protein 4.1 containing resealed ghostssubjected to increased shear stress. Arduini et al., Life Sci. 47:2395-2400 (1990). Thus, LC may exert a stabilizing effect of themembrane through a spectrin interaction with one or more cytoskeletalcomponents. A recent electron paramagnetic resonance study ofButterfield and Rangachari, Life Sci. 52: 297-303 (1992), on the redcell spectrin-actin interaction showed that LC significantly reduced thesegmental motion of spin-labeled sties on spectrin, confirming theprevious suggestion of an involvement of LC in strengthening theinteraction between spectrin and actin, Arduini et al., supra.

In addition to a potential biophysical action described above, theimprovements observed in the LC-stored red cells may also be the resultof a favorable metabolic process. Normally, the deacylation-reacylationcycle of membrane phospholipids requires ATP for generation of acyl-CoA.The acyl moiety of acyl-CoA is then transferred into lysophospholipidsby lysophospholipid acyl-CoA transferase. In addition, during anoxidative challenge, the membrane repair process of RBC phospholipidsfollows the same metabolic pathway. Recent findings have shown that CPTaffect the recylation process of membrane phospholipids in red cells andneuronal cells by modulating the size of the acyl-CoA pool between theactivation step of the fatty acid and its transfer intolysophospholipids. Arduini et al., J. Biol. Chem. 267: 12673-12681(1992). In addition, pulse-chase and ATP depletion studies havedemonstrated that the red cell acylcarnitine pool serves as a reservoirof activated acyl groups at no cost of ATP. Arduini et al., Biochem.Biophys. Res. Comm. 187: 353-358 (1994).

The enhancement of radioactive palmitate incorporation into membrane PEof red cells stored in AS-3 alone suggests that long term storage (witha progressive RBC ATP depletion) causes an increased demand of activatedacyl units for the recylation of membrane phospholipids (FIG. 4). Duringthe first five weeks of storage red cells preserved in AS-3 without LCseem to incorporate more palmitate into PE than red cells stored in AS-3containing LC (FIG. 4). Red cells stored in the presence of LC were ableto incorporate more palmitate into PE at the end of the storage. Thisfinding may suggest that an oxidative challenge is somehow operative,since the exposure of red cells to oxidant strongly stimulates themembrane PE recylation process, but not that of membrane PC. Dise etal., Biochem. Biophys. Acta 859: 69-78 (1986). Of interest, during thefirst five weeks of storage red cells preserved in AS-3 alone seem toincorporate more palmitate into PE than red cells stored in AS-3containing LC (FIG. 4). This suggests that in the latter case CPT maycompete with the reacylating enzyme for acyl-CoA utilization. Inagreement with this concept, however, we should have observed a muchgreater difference at the end of the storage period, when the acyl-CoArequirement for the reacylation process is the highest. This was not thecase. Red cell stored with or without LC showed a similar incorporationrate at the end of the storage (FIG. 4). In addition, we have shown thatred cell CPT, under a variety of different experimental conditions, doesnot compete with the reacylation of membrane phospholipids. Arudini etal., Life Chem. Rep. 12: 49-54 (1994).

Radioactive PLC formation in the LC-supplemented red cells is greaterthan that found in red cell stored without LC (FIG. 3). In addition, thetime course of PLC formation in red cells stored with LC showed aninteresting bell shape curve with a nadir at the third week of storage.The radioactive PLC formation is reflective of the pool size of coldlong-chain acylcarnitine and the direction of CPT-mediated acyl flux inintact red cells. During the first three weeks of storage withrelatively high glycolytic activity and APT availability, CPT seems todrive the acyl flux toward acylcarnitine in red cells stored in thepresence of LC (FIG. 5 a). After the third week of storage, with LCpresent the flux is then reversed. These changes may reflect essentiallythe ability of CPT to buffer activated acyl-units: an increasedrequirement of acyl-CoA for the reacylation process results in a lowerproduction of PLC and vice versa, and may represent a mechanism whereCPT is used to buffer the increase of demand of acyl units for thereacylation process (FIG. 5 b).

The higher 24-hour % recovery and circulating lifespan represents animprovement of approximately 15% in terms of potency (amount oftransfused circulating RBC available to the recipient times averagelifespan). This increase in potency could translate clinically into areduction in transfusion requirements in chronically transfused patientssuch as in thalassemics or in patients with bone marrow failure.Alternatively, it may be possible to extend the shelf-life of liquidstored RBCs.

Our findings suggest that the presence of LC in the preservation mediumduring RBC storage may have a sparing action on the ATP pool used by thereacylation of phospholipids for membrane repair. This favorablemetabolic process, associated with a possible beneficial biophysicalaction, may thus explain the reduced hemolysis, higher ATP levels, andthe improved in vivo recovery and survival of the LC-stored red cells.

IRRADIATION

The problem of contamination of blood products, including whole blood,RBC, PC and the like, can be reduced by substantial amount byirradiation. Levels of irradiation necessary for sterilization, andsubstantially 100% mortality of microbiological agents, have been widelyexplored. Additionally, more importantly, leukocytes may be destroyed bysimilar irradiation. A variety of types of irradiation can be used,including gamma radiation (Cobalt G G, Van de Graf acceleration), UVirradiation, red light irradiation, etc. A close equivalent to about20-50 cG gamma irradiation is sufficient.

Worldwide, between 60 and 80 million units of whole blood are collectedannually and used in the transfusion support of a variety of patientpopulations. In the underdeveloped countries collection rates per 1000population are lower and most blood transfusions are given in thetreatment of obstetrical and pediatric cases, particularly malariaassociated anemia. In the developed countries, collection rates per 1000population are 50-10 times higher and most transfusions are given insurgery (50%) or in the treatment of patients with cancer associatedanemia, bone marrow transplantation, non-malignant gastrointestinalbleeding (FIG. 1). There are many potential adverse effects associatedwith the transfusion of allogenic blood. One particular complicationadversely associated with blood transfusion is the rare and usuallyfatal entity known as Transfusion associated graft versus host disease(TA-GVHD), a complication mediated by viable allogenic immunocytes.

TA-GVHD disease is a rare complication of blood transfusion potentiallyseen in two types of blood transfusion recipient patient populations.TA-GVHD has a mortality approaching 100% and prevention is the onlyeffective approach at this time. First, in immunocompromised patients,such as patients after bone marrow or other organ transplantation,Hodgkins disease or hereditary deficiencies of the immune system.Second, in nonimmunocompromised patients, when HLA similarity existsbetween blood donor and blood recipient. This is most often seen indirected donations from close relatives or in populations of morelimited HLA polymorphism such as in Japan and Israel. On account ofthis, it is universal practice to irradiate cellular blood products withgamma irradiation to a mid-plane dose of approximately 25 centigray (cG)in order to destroy the replicating ability of viable immunocytes. Itshould be noted that TA-GVHD is associated with cellular products whichare fresh, i.e. generally less than 15 days. However, “aging” of bloodis not as yet an accepted practice in preventing this complication.Although the immunocytes are part of the allogenic leukocyte population,the degree of leukodepletion currently achieved with third generationfilters is not considered currently adequate to prevent thiscomplication. Thus, gamma radiation at this time remains the onlyaccepted prophylactic intervention.

The difficulty with gamma irradiation of red cells in particular is thepotential to damage the cell membrane. It is clear that irradiation tothis dose produces a loss in potency of approximately 7-8% as measuredin vitro by a reduction in red cell ATP, increased hemolysis, andincreased supernatant potassium. These changes are consistent with amembrane damage effect. These in vitro changes are associated with areduction in the 24 hour recovery of gamma irradiation red blood cells.With regard to platelet products, at least one publication has suggestedsome loss in viability.

Considerable evidence indicates that gamma irradiation exerts itseffects by generating activated oxygen species, such as singlet oxygen,hydroxy, radical, and superoxide anion. These species induceintracellular damage to DNA, thus prevent cell replication, aprerequisite to TAGVHD. However, these same oxygen species may oxidizemembrane lipids on the red cell and possibly platelet membrane, inducinga membrane lesion which reduces the quality (potency) of the cellularproduct.

LC is known to play a key role in the transportation of long chain fattyacids across the mitochondrial membrane. Hereditary disorders in whichthere is a failure of the carnitine system to transport long chain fattyacids results in significant impairment in skeletal muscle function.Recently, there has been increasing interest in the role of LC in redcell membrane. What has been surprising, however, is that red cells lackmitochondria, and thus, considerable curiosity surrounding the presenceof LC and carnitine palmitoyl transferase, an enzyme involved inreversible acylation of LC. It was at first unclear as to the role whichthese might play within red cell. The red cell may be subjected tooxidant stress throughout it's long life cycle in vivo, and repair ofoxidized membrane lipids involving LC could be important for the normalsurvival of red cells.

Early increase in acylated carnitine during a time of increased ATPavailability may function as a reservoir of activated fatty acids, whichcan subsequently be used in a repair mechanism for damaged oxidizedmembranes lipids. Such an explanation would well explain the reducedhemolysis observed during the in vitro storage of red blood cells supraand in addition, would explain the improvement observed with in vivorecovery and survival. The net effect of LC addition is an approximate17% increase in potency.

Accordingly, LC may be used to abrogate or prevent membrane lesionsinduced by irradiation. This would occur through the ability ofcarnitine stored in red cells to repair oxidized membrane lipids invitro.

To limit blood products (RCC, PC and the like) susceptibility tomembrane lesions and hemolysis, the blood product may be first suspendedin a solution including LC in an amount of 0.25 mM-50 mM, cell membranemaintenance and suppression of hemolysis is achieved to a sufficientdegree that the sealed product can be irradiated for the purposes ofsterilization, and subsequently may enjoy an extended shelf life andcirculation half-life after transfusion. Viability on the order ofcurrent viabilities can be achieved, with materials more nearly certainto be sterile and unlikely to introduce TA-GVHD, due to irradiationafter sealing the blood product suspension. It it to be emphasized thatthe term blood product, in this connection, is to be interpretedbroadly, to include whole blood, blood plasma, RCCs, PCs, mixtures andthe like.

The invention of this patent application has been disclosed in bothgeneric terms and by reference to specific examples. Variations willoccur to those of ordinary skill in the art without the exercise ofinventive faculty. In particular, alternate stabilizing compositions,blood products, preservatives, inhibitors and the like may be modified,without the exercise of inventive skill. Additionally, specific levels,viability periods and circulation half-lifes will vary from donor todonor, and recipient and recipient. Such variations remain within thescope of the invention, unless specifically excluded by the recitationsof the claims set forth below.

TABLE 1 RBC Pre-Storage Characteristics of the Red Cell concentratesTest Control (L-carnitine) Unit Volume (mL) 305 ± 38  295 ± 41  Unit Hct(%) 60 ± 3  60 ± 3  Unit WBC (×10⁹) 2.6 ± 1.1 2.4 ± 1.1 ATP (μmol/g Hb)4.6 ± 0.2 4.3 ± 0.4 Supernatant Hb (mg/dL) 34 ± 15 26 ± 8  SupernatantK + (mEq/L) 2.3 ± 0.2 2.2 ± 0.3 Osmotic Fragility (%) 50 ± 4  49 ± 4 

TABLE 2 Post-Storage (42 days) Characteristics of the Red Cellconcentrates Test Control (L-Carnitine) In Vitro parameters Glucose(mg/dL) 208 ± 33  193 ± 40  Lactate (mg/dL) 201 ± 27  199 ± 37  pH 6.33± 0.03 6.32 ± 0.04 ATP (μmol/g Hb) 3.01 ± 0.42  3.24 ± 0.38* Hemolysis(%) 0.47 ± 0.41  0.30 ± 0.22* Supernatant K + (mEq/L) 61 ± 4  60 ± 3 Osmotic Fragility (%) 51 ± 3  50 ± 4  Morphological Score 69 ± 8  68 ±15 In Vivo parameters 24 H % Recovery (single label) 81.1 ± 6.2  84.0 ±4.4  24 H % Recovery (double label) 80.1 ± 6.0  83.9 ± 5.0* RBC Mass(mL) 1634 ± 510  1591 ± 534  Survival (days) 85.9 ± 14.3  96.1 ± 11.2**(p < 0.05)

1. A method for preparing a blood product concentrate for storage,comprising: (1) suspending said blood product concentrate in a supportsolution, to obtain a suspension of said blood product concentrate; and(2) irradiating said suspended blood product concentrate, to effectsterilization and inactivation of leukocytes, to obtain a irradiated,suspended blood product concentrate, wherein: said blood productconcentrate is a platelet concentrate; and said support solutionconsists essentially of: (1) a compound selected from the groupconsisting of L-carnitine, alkanoyl L-carnitines, pharmaceuticallyacceptable salts thereof, and mixtures thereof; (2) water; (3) a buffer;and (4) a carbohydrate, and wherein said compound is present in saidsupport solution in an amount effective to maintain the membrane of orplatelets present in said irradiated, suspended blood productconcentrate.
 2. The method of claim 1, wherein said compound is presentin said support solution in a concentration of 0.25 to 50 mM.
 3. Themethod of claim 1, wherein said compound is L-carnitine.
 4. The methodof claim 1, wherein said compound is L-carnitine and wherein saidcompound is present in said support solution in a concentration of 0.25to 50 mM.
 5. The method of claim 1, wherein said compound is selectedfrom the group consisting of acetyl L-carnitine, propionyl L-carnitine,butyryl L-carnitine, isobutyryl L-carnitine, valeryl L-carnitine, andisovaleryl L-carnitine.
 6. The method of claim 1, wherein said compoundis selected from the group consisting of acetyl L-carnitine, propionylL-carnitine, butyryl L-carnitine, isobutyryl L-carnitine, valerylL-carnitine, and isovaleryl L-carnitine and wherein said compound ispresent in said support solution in a concentration of 0.25 to 50 mM. 7.The method of claim 1, wherein said irradiating is irradiating withgamma-irradiation.
 8. The method of claim 1, wherein said buffer isselected from the group consisting of ACED, CPD, and CPD2/A3.
 9. Themethod of claim 1, wherein said support solution further contains a saltselected from the group consisting of phosphate salts, citrate salts,and other balancing salts.
 10. The method of claim 1, wherein saidsupport solution is an artificial plasma.
 11. A method for preparing ablood product concentrate for storage, comprising: (1) suspending saidblood product concentrate in a support solution, to obtain a suspensionof said blood product concentrate; and (2) irradiating said suspendedblood product concentrate, to effect sterilization and inactivation ofleukocytes, to obtain an irradiated, suspended blood productconcentrate, wherein: said blood product concentrate is a and plateletconcentrate; and said support solution consists of: (1) a compoundselected from the group consisting of L-carnitine, alkanoylL-carnitines, pharmaceutically acceptable salts thereof, and mixturesthereof; (2) water; (3) a buffer; and (4) a carbohydrate, and whereinsaid compound is present in said support solution in an amount effectiveto maintain the membrane of or platelets present in said irradiated,suspended blood product concentrate.
 12. The method of claim 11, whereinsaid compound is present in said support solution in a concentration of0.25 to 50 mM.
 13. The method of claim 11, wherein said compound isL-carnitine.
 14. The method of claim 11, wherein said compound isL-carnitine and wherein said compound is present in said supportsolution in a concentration of 0.25 to 50 mM.
 15. The method of claim11, wherein said compound is selected from the group consisting ofacetyl L-carnitine, propionyl L-carnitine, butyryl L-carnitine,isobutyryl L-carnitine, valeryl L-carnitine, and isovaleryl L-carnitine.16. The method of claim 11, wherein said compound is selected from thegroup consisting of acetyl L-carnitine, propionyl L-carnitine, butyrylL-carnitine, isobutyryl L-carnitine, valeryl L-carnitine, and isovalerylL-carnitine and wherein said compound is present in said supportsolution in a concentration of 0.25 to 50 mM.
 17. The method of claim11, wherein said irradiating is irradiating with gamma-irradiation.